High-Performance Liquid Chromatographic Analysis of Dipyrone Metabolites to Study Their Formation in Human Liver Microsomes

Pharmaceutical Research -     

The Effect of RPR 102341 on Theophylline Metabolism and Phenacetin O-Deethylase Activity in Human Liver Microsomes

Purpose. RPR 102341 is structurally similar to the fluoroquinolone class of antibiotics. Because some fluoroquinolones have been shown to inhibit theophylline metabolism, concomitant administration may increase plasma levels of theophylline resulting in serious adverse effects. The purpose of this study was to determine if RPR 102341 affects theophylline metabolism in vitro and, thus, predict whether a clinically significant drug interaction is likely to occur. In addition, the effect of RPR 102341 on phenacetin O-deethylase activity was determined to address the enzymatic basis of a potential drug interaction. Methods. The in vitro theophylline metabolism assay was conducted according to a modification of a published procedure. The phenacetin O-deethylase assay was conducted according to a modification of a published procedure. Results. The rate of conversion of theophylline to 3-methylxanthine in human liver microsomes in the presence of 100 M and 500 M RPR 102341 was 93.6 and 106 percent of the control reactions, respectively. The formation of 1-methylxanthine was 97.6 and 100 percent of the control, and 1,3-dimethyluric acid formation was 88.9 and 95.2 percent of control at 100 M and 500 M RPR 102341, respectively. In agreement, RPR 102341 caused no inhibition of human liver CYP1A2—catalyzed phenacetin O-deethylase activity. Finally, no inhibition was observed when RPR 102341 was incubated with human liver microsomes and an NADPH regenerating system prior to the addition of theophylline. Conclusions. Based on these studies, RPR 102341 is not expected to cause significant drug interactions with theophylline.     

大鼠肝微粒体代谢研究槲皮素对CYP1A2,CYP2E1,CYP3A2活性的影响及抑制机制

目的体外代谢研究槲皮素对大鼠肝CYP1A2,CYP2E1,和CYP3A2活性的影响。研究其抑制强度及抑制机制。方法QU与底物共同温孵,HPLC检测底物特定的代谢产物生成量的变化反映对应亚酶的活性变化。比较槲皮素与酮康唑,红霉素在相同浓度下对CYP3A2的抑制能力强弱。不同浓度槲皮素对CYP3A2和CYP2E1底物代谢产物生成双倒数直线的影响初步分析槲皮素可能的抑制机制。结果各HPLC检测方法线性相关系数均>0.9991,RSD均<8.4%,回收率91.1%-107.6%。槲皮素在体外0～8μmol·L-1诱导大鼠肝微粒体CYP1A2的活性达338.1%,并抑制CYP2E1(49.2%),和CYP3A2(60.3%)。槲皮素对CYP3A2的抑制能力在酮康唑和红霉素之间。槲皮素竞争性抑制CYP3A2右美沙芬N脱甲基反应,非竞争性抑制CYP2E1氯唑沙宗6羟化反应。结论槲皮素对多个CYP450亚酶有抑制作用,它是有效的CYP3A竞争性抑制剂。做为黄酮类植物雌激素,槲皮素有分子结构的优势亦有对CYP450酶调控能力而具有未来抗肿瘤药物研究的潜力。     

Comparative Metabolism of Debrisoquine, 7–Ethoxyresorufin and Benzo(a)pyrene in Liver Microsomes from Humans,and from Rats Treated with Cytochrome P–450 Inducers

Abstract: The metabolism of debrisoquine, 7–ethoxyresorufin and benzo(α)pyrene has been studied in human liver microsomes. There was a significant correlation (r = 0.70, P < 0.05) between debrisoquine hydroxylation and 7–ethoxyresorufin 0–deethylation among various livers, and debrisoquine inhibited 7–ethoxyresorufin deethylation competitively. These results suggest that debrisoquine and 7–ethoxyresorufin may be metabolised by a common P–450 form in human liver. The effect of cytochrome P–450 inducers on the metabolism of the three substrates was also examined in rat liver. Debrisoquine hydroxylation was not enhanced by phenobarbitone, β–naphthoflavone or isosafrole     

肝细胞微粒体的制备和细胞色素P450氧化酶活性测定

目的：为测定人肝细胞微粒体细胞色素Ｐ４５０氧化酶的活性。方法：用差速离心法制备３例人肝细胞微粒体。结果：细胞色素Ｐ４５０的含量为０．５２３±０．００５ｎｍｏｌ·ｍｇ－１；细胞色素ｂ５为０．２８５±０．０２５ｎｍｏｌ·ｍｇ－１；氨基比林Ｎ－脱甲基酶的活力为０．５±０．６ｎｍｏｌ·ｍｇ－１；乙基吗啡Ｎ－脱甲基酶活力为０．９８±０．０８ｎｍｏｌ·ｍｇ－１。结论：Ｐ４５０酶活性影响因素较多，个体差异大。临床用药时应考虑患者的个体情况。     

Correlation between Changes in Enzymatic Activities and Induction of Different Forms of Rat Liver Microsomal Cytochrome P-450 after Phenobarbital-, 3-Methylcholanthrene- and 16α-Cyanopregnenolone Treatment

Abstract Multiple forms of liver microsomal cytochrome P-450 in rats were identified on SDS-polyacrylamide gels stained for protein and peroxidase activity after induction with phenobarbital, 3-methylcholanthrene, and 16α-cyanopregnenolone. The induced forms were correlated to the in vitro metabolism of biphenyl, benzo(a)pyrene and the steroids 4-androstene-3,17-dione and 5α-androstane-3α,17β-diol. Induction of two forms with apparent molecular weights of 54,000 (RLvMc P-450₅₄) and 50,000 (RLvMc P-45 0₅₀) was obtained with phenobarbital, induction of RLvMc P-450₅₅ and RLvMc P-450₅₈ with 3-methylcholanthrene and induction of RLvMc P-450₅₄ with 16α-cyanopregnenolone. The RLvMc P-450₅₀ was mainly associated with the formation of benzo(a)pyrene-4,5-dihydrodiol, and 7α-hydroxy-4-androstene-3,17-dione. The RLvMc P-450₅₅ and/or the RLvMc P-450₅₈ was mainly associated with the formation of 2- and 3-hydroxybiphenyl and benzo(a)pyrene-7,8-dihydrodiol and RLvMc P-450₅₄ was to some extent associated with the formation of benzo(a)pyrene-4,5-dihydrodiol and several metabolites of 5α-androstane-3α, 17β-diol. It is suggested that SDS-polyacrylamide gel electrophoresis may be a valuable complement to enzyme assays in evaluating effects of drugs and environmental chemicals on the liver microsomal hydroxylase system.     

Dose Dependent Induction of Rat Liver Microsomal Cytochrome P-450 and Microsomal Enzymatic Activities after Inhalation of Toluene and Dichloromethane

Abstract: Sprague-Dawley rats were exposed, by inhalation, to toluene and dichloromethane (500, 1,500 or 3,000 p.p.m.) and to benzene (1,500 p.p.m.) for three days. Toluene and benzene increased the concentration of liver microsomal cytochrome P-450. A dose dependent increase in the in vitro liver microsomal formation of several metabolites of biphenyl and benzo(a)pyrene was observed for both dichloromethane and toluene. At the highest dose-level the increase in the in vitro formation of benzo(a)pyrene-7,8-dihydrodiol was more than threefold for both dichloromethane and toluene whereas the formation of benzo(a)pyrene-4,5-dihydrodiol increased more than five-fold following exposure to toluene but less than two-fold after exposure to dichloromethane. Our results suggest that dichloromethane and toluene can modify the metabolism and thereby the toxicity of other environmental contaminants.     

The Possible Role of Cytochrome P-450 in the Liver “First Pass Elimination” of a P-Receptor Blocking Drug

The highly lipid soluble β-receptor blocking drug alprenolol interacts with high affinity with rat liver microsomal cytochrome P-450, is rapidly metabolized in the liver and exhibits a marked liver “first pass elimination” (FPE) in the rat. It thus has a low oral bioavailability in this species. In order to investigate the possible role of the cytochrome P-450 system in the FPE we studied the influence of the three P-450 inhibitors SKF 525-A, imipramine and metyrapone and of phenobarbital treatment on the disposition kinetics of alprenolol in a series of experimental models. Alprenolol rapidly gave rise to a type I spectral change on addition to intact liver cells, indicating a rapid hepatic uptake. The maximal magnitude of this spectrum increased about twofold after phenobarbital treatment of rats in both microsomes and isolated liver cells. Imipramine, SKF 525-A and metyrapone partly displaced ³H-al-prenolol from non-metabolizing partly purified cytochrome P-450 and liver cell preparations (20°). Imipramine and SKF 525-A were about equally effective in this respect whereas metyrapone was much less potent. At 37° the metabolism of alprenolol was rapid and of about similar activity (per nmoles of cytochrome) in liver microsomes, isolated liver cells and in the perfused liver (at a high dose). The K_m-value was similar in microsomes and in isolated liver cells. A similar metabolic inhibitory pattern was found in microsomes and isolated liver cells. SKF 525-A was the most efficient inhibitor followed by imipramine and then metyrapone. The same inhibitory pattern was found for the hepatic extraction of alprenolol. Moreover, the hepatic extraction of alprenolol was dose dependent. Imipramine in a high dose increased the area under the blood concentration curve by a factor of ten after oral administration of alprenolol in the conscious rat. The above findings suggest that cytochrome P-450 is, at least partly, responsible for the degree of hepatic extraction and metabolism (FPE) of alprenolol. This view was also supported by the findings that the perfused liver showed an increased capacity for the extraction and metabolism of alprenolol after phenobarbital treatment. The cytochrome P-450 system may influence the hepatic extraction of alprenolol in rat liver by providing an intracellular “high affinity binding pool” for the unchanged drug. The subsequent metabolic step seems to be important since it “unloads” P-450 so that it can bind new drug molecules.     

The time-dependent effects of St John’s wort on cytochrome P450, uridine diphosphate-glucuronosyltransferase,glutathione S-transferase,and NAD(P)H-quinone oxidoreductase in mice

Hypericum perforatum [St. John’s wort (SJW)] is known to cause a drug interaction with the substrates of cytochrome P450 (P450, CYP) isoforms, mainly CYP3A. This study aims to determine the dose response and time course of the effects of SJW extract on P450s, UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), and NAD(P)H-quinone oxidoreductase (NQO) in mice. The oral administration of SJW extract to male mice at 0.6 g/kg/d for 21 days increased hepatic oxidation activity toward a Cyp3a substrate nifedipine. By extending the SJW treatment to 28 days, hepatic nifedipine oxidation (NFO) and warfarin 7-hydroxylation (WOH) (Cyp2c) activities were increased by 95% and 34%, respectively. Immunoblot analysis of liver microsomal proteins revealed that the Cyp2c protein level was elevated by the 28-day treatment. However, the liver microsomal activities of the oxidation of the respective substrates of Cyp1a, Cyp2a, Cyp2b, Cyp2d, and Cyp2e1 remained unchanged. In the kidney, SJW increased the NFO, but not the WOH activity. The extended 28-day treatment did not alter mouse hepatic and renal UGT, GST, and NQO activities. These findings demonstrate that SJW stimulates hepatic and renal Cyp3a activity and hepatic Cyp2c activity and expression. The induction of hepatic Cyp2c requires repeated treatment for a period longer than the initial induction of Cyp3a.     

Stoichiometric Interaction of a β-Receptor Blocking Drug with Fraction of Liver Cytochrome P-450 as a Possible Cause for its Dose-dependent Availability

Abstract The liver's ability to bind and metabolize “first-pass” drugs is a major factor responsible for their blood concentrations on oral administration. Therefore we have characterized the binding of such a drug, the adrenergic β-receptor antagonist alprenolol, to rat liver. The microsomal fraction accounted for almost all the liver binding capacity. On titration of microsomes with alprenolol, the induced type I spectral change, reflecting alprenolol-cytochrome P-450 interaction, showed a high and a low affinity binding phase with identical “optical capacity”. The spectral dissociation constant for the high affinity binding was 0.17 μM. By use of spectral methods we found that 13–14 per cent of the total microsomal cytochrome P-450 participated in the alprenolol binding at the end of the first phase and 27 per cent at the end of the second phase. Spectral titrations showed that SKF 525-A and imipramine interact with the same sites of cytochrome P-450 as alprenolol. Binding studies with radioactive alprenolol showed the presence of a minimum of three classes of microsomal binding sites. The last had a very high capacity. The first phase caused a marked non-linearity in the overall microsomal binding of the drug, and it had almost identical capacity (0.12 mol alprenolol/mol P-450) and affinity (K_s = 0.21 μM) as the first type I spectral binding phase. This strongly indicates that this first “overall” binding phase reflects selective binding to P-450, which is further strengthened by the fact that SKF 525-A and imipramine markedly influenced this phase. Moreover, this phase was not present in plasma membranes, which do not contain cytochrome P-450. Hence we conclude that a stoichiometric (1:1) interaction of alprenolol with a 12–14 per cent fraction of rat liver microsomal cytochrome P-450 causes a non-linear overall microsomal binding. Such a phenomenon probably explains this drug's dose-dependent availability. We suggest that when this high affinity site is saturated the low affinity site of cytochrome P-450 oxidizes a large fraction of the alprenolol escaping ‘first pass’ liver extraction.     

Metabolism of the tryptamine‐derived new psychoactive substances 5‐MeO‐2‐Me‐DALT, 5‐MeO‐2‐Me‐ALCHT,and 5‐MeO‐2‐Me‐DIPT and their detectability in urine studied by GC–MS,LC–MSn,and LC‐HR‐MS/MS

Many N,N‐dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5‐methoxy‐2‐methyl‐N,N‐diallyltryptamine (5‐MeO‐2‐Me‐DALT), 5‐methoxy‐2‐methyl‐N‐allyl‐N‐cyclohexyltryptamine (5‐MeO‐2‐Me‐ALCHT), and 5‐methoxy‐2‐methyl‐N,N‐diisopropyltryptamine (5‐MeO‐2‐Me‐DIPT) using gas chromatography–mass spectrometry (GC–MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC–MSⁿ), and liquid chromatography‐high‐resolution tandem mass spectrometry (LC‐HR‐MS/MS). For metabolism studies, urine was collected over a 24 h period after administration of the compounds to male Wistar rats at 20 mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC‐HR‐MS/MS. 5‐MeO‐2‐Me‐DALT (24 phase I and 12 phase II metabolites), 5‐MeO‐2‐Me‐ALCHT (24 phase I and 14 phase II metabolites), and 5‐MeO‐2‐Me‐DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O‐demethylation, hydroxylation, N‐dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O‐demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N‐dealkylation. For SUSAs, GC–MS, LC‐MSⁿ, and LC‐HR‐MS/MS were applied to rat urine samples after 1 or 0.1 mg/kg BW doses, respectively. In contrast to the GC–MS SUSA, both LC–MS SUSAs were able to detect an intake of 5‐MeO‐2‐Me‐ALCHT and 5‐MeO‐2‐Me‐DIPT via their metabolites following 1 mg/kg BW administrations and 5‐MeO‐2‐Me‐DALT following 0.1 mg/kg BW dosage. Copyright © 2017 John Wiley & Sons, Ltd.     

Contribution of cytochrome P450 3A4 and 3A5 to the metabolism of atorvastatin

Atorvastatin is a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor that is mainly metabolized by cytochrome P450 (CYP) 3A4. A recent study showed that the lipid-lowering effect of statins is affected by the CYP3A5 polymorphism. Therefore, it was investigated whether CYP3A5 contributes to the metabolism of atorvastatin. Two metabolites of atorvastatin, para- and ortho-hydroxyatorvastatin, were produced by human liver microsomes and human recombinant CYP3A enzymes, and the enzyme kinetic pattern exhibited substrate inhibition. The intrinsic clearance (CL_int) rates of para- and ortho-hydroxyatorvastatin by CYP3A4 were 2.4- and 5.0-fold of the respective CL_int rates of CYP3A5, indicating that CYP3A4 is the major P450 isoform responsible for atorvastatin metabolism. These results suggest that atorvastatin is preferentially metabolized by CYP3A4 rather than by CYP3A5, and thus the genetic CYP3A5 polymorphism might not be an important factor in the inter-individual variation of atorvastatin disposition and pharmacodynamics in human.     

Effect of Serum, α-1 Acid Glycoprotein,Lipoproteins and Albumin on Human Mononuclear Leucocyte β-Adrenoceptors

Abstract: The effects of serum, α-1 acid glycoprotein (AAG), serum lipoproteins (SLP) and human serum albumin (HSA) on ³H-(-)-dihydroalprenolol (³H-(-)-DHA) binding and (-)-isoproterenol ((-)-IPR) induced cyclic AMP (cAMP) elevation in human peripheral blood mononuclear leucocytes (MNL) were investigated. The saturable binding of ³H-(-)-DHA was decomposed into two classes of binding sites with maximum binding capacity of approximately 1400 and 30000 sites/cell and with dissociation constants (K_d) of approximately 0.7 and 65 nM. Stimulation of the MNL β-adrenoceptors by (-)-IPR caused a concentration dependent cAMP accumulation (EC50 ～0.2 μM) with maximum level approximately 250% above basal. For all single leucocyte preparations, 30–35 min. exposure to serum, AAG and SLP increased the number of β-adrenoceptors with 100–200% and the maximal responsiveness to (-)-IPR with 30–90%. The presence of proteins did not change the K_d or the EC50. (-)-Alprenolol inhibited concentration dependently the serum induced increment in (-)-IPR-responsiveness. Serum, AAG and SLP did also increase the number of low affinity binding sites with 25–40% without effect on the K_d. HSA had no consistent effect on β-adrenergic binding or stimulation. The present study shows that serum, AAG and SLP influence the number and function of MNL β-adrenoceptors in vitro.     

Synthesis,Hypoglycaemic, Hypolipidemic and PPARγ Agonist Activities of 5‐(2‐Alkyl/aryl‐6‐Arylimidazo[2,1‐b][1,3,4]thiadiazol‐5‐yl)methylene‐1,3‐Thiazolidinediones

A novel series of 5‐(2‐alkyl/aryl‐6‐arylimidazo[2,1‐b][1,3,4]thiadiazol‐5‐yl)methylene‐1,3‐thiazolidinediones were synthesized as possible PPARγ agonists. The structures of these target molecules were established by spectral and analytical data. All the newly synthesized compounds were screened for their in vivo hypoglycaemic and hypolipidemic activity in male Wistar rats. Further, compounds with good activity were screened for PPARγ agonist activity. Among the screened compounds, 5‐{[2‐Cyclohexyl‐6‐(4‐methoxyphenyl)imidazo[2,1‐b] [1,3,4]thiadiazol‐5‐yl]methylene}‐1,3‐thiazolidine‐2,4‐dione (3i) exhibits promising hypoglycaemic and hypolipidemic activity via potential PPARγ agonist activity.     

Functional properties and substrate characterization of human CYP26A1, CYP26B1, and CYP26C1 expressed by recombinant baculovirus in insect cells

Introduction

The cytochrome P450 CYP26 family of retinoic acid (RA) metabolizing enzymes, comprising CYP26A1, CYP26B1, and CYP26C1 is critical for establishing patterns of RA distribution during embryonic development and retinoid homeostasis in the adult. All three members of this family can metabolize all trans-RA. CYP26C1 has also been shown to efficiently metabolize the 9-cis isomer of RA.

Methods

We have co-expressed each of the CYP26 enzymes along with the NADPH-cytochrome P450 oxidoreductase using a baculovirus/Sf9 insect cell expression system to determine the enzymatic activities of these enzymes in cell free preparations and have established an in vitro binding assay to permit comparison of binding affinities of the three CYP26 enzymes.

Results

We demonstrated that the expressed enzymes can efficiently coordinate heme, as verified by spectral-difference analysis. All CYP26s efficiently metabolized all-trans-RA to polar aqueous-soluble metabolites, and in competition experiments exhibited IC₅₀ values of 16, 27, and 15 nM for CYP26A1, B1, and C1 respectively for all-trans-RA. Furthermore, this metabolism was blocked with the CYP inhibitor ketoconazole. CYP26C1 metabolism of all trans-RA could also be effectively competed with 9-cis RA, with IC₅₀ of 62 nM, and was sensitive to ketoconazole inhibition.

Discussion

CYP26 enzymes are functionally expressed in microsomal fractions of insect cells and stably bind radiolabeled RA isomers with affinities respecting their substrate specificities. We demonstrated that compared to CYP26A and CYP26B, only CYP26C1 was able to bind with high affinity to 9-cis-RA. These assays will be useful for the screening of synthetic substrates and inhibitors of CYP26 enzymes and may be applicable to other cytochrome P450s and their respective substrates.     

Reliability and sensitivity of performance tests in human psychopharmacology. A commentary on the articles by parrott (1991), human psychopharmacology, 6, 1–9, 91–98, 197–207

This commentary argues that the conventional assessment of the reliability of psychological tests may be inappropriate in psychopharmacology. The conventional approach depends upon the assessment of differences between individuals rather than changes within individuals. It thus favours measures of trait, while those required for investigating drug-induced changes are measures of state. The sensitivity of a measure to change is a more useful criterion for inclusion in a test battery for the investigation of the effects of drugs.     

Study of the in vitro and in vivo metabolism of the tryptamine 5‐MeO‐MiPT using human liver microsomes and real case samples

The synthetic tryptamine 5‐methoxy‐N‐methyl‐N‐isopropyltryptamine (5‐MeO‐MiPT) has recently been abused as a hallucinogenic drug in Germany and Switzerland. This study presents a case of 5‐MeO‐MiPT intoxication and the structural elucidation of metabolites in pooled human liver microsomes (pHLM), blood, and urine. Microsomal incubation experiments were performed using pHLM to detect and identify in vitro metabolites. In August 2016, the police encountered a naked man, agitated and with aggressive behavior on the street. Blood and urine samples were taken at the hospital and his premises were searched. The obtained blood and urine samples were analyzed for in vivo metabolites of 5‐MeO‐MiPT using liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS). The confiscated pills and powder samples were qualitatively analyzed using Fourier transform infrared (FTIR), gas chromatography–mass spectrometry (GC–MS), LC‐HRMS/MS, and nuclear magnetic resonance (NMR). 5‐MeO‐MiPT was identified in 2 of the seized powder samples. General unknown screening detected cocaine, cocaethylene, methylphenidate, ritalinic acid, and 5‐MeO‐MiPT in urine. Seven different in vitro phase I metabolites of 5‐MeO‐MiPT were identified. In the forensic case samples, 4 phase I metabolites could be identified in blood and 7 in urine. The 5 most abundant metabolites were formed by demethylation and hydroxylation of the parent compound. 5‐MeO‐MiPT concentrations in the blood and urine sample were found to be 160 ng/mL and 3380 ng/mL, respectively. Based on the results of this study we recommend metabolites 5‐methoxy‐N‐isopropyltryptamine (5‐MeO‐NiPT), 5‐hydroxy‐N‐methyl‐N‐isopropyltryptamine (5‐OH‐MiPT), 5‐methoxy‐N‐methyl‐N‐isopropyltryptamine‐N‐oxide (5‐MeO‐MiPT‐N‐oxide), and hydroxy‐5‐methoxy‐N‐methyl‐N‐isopropyltryptamine (OH‐5‐MeO‐MiPT) as biomarkers for the development of new methods for the detection of 5‐MeO‐MiPT consumption, as they have been present in both blood and urine samples.     

An Interactive Human Carbonic Anhydrase‐II (hCA‐II) Receptor – Pharmacophore Molecular Model & Anti‐Convulsant Activity of the Designed and Synthesized 5‐Amino‐1,3,4‐Thiadiazole‐2‐Thiol Conjugated Imine Derivatives

New imines, derived from aromatic aldehyde, chalcones and 5‐amino‐1,3,4‐thiadiazole‐2‐thiol exhibited promising anti‐convulsant activity which is explained through chemo‐biological interactions at receptor site producing the inhibition of human Carbonic Anhydrase‐II enzyme (hCA‐II) through the proposed pharmacophore model at molecular levels as basis for pharmacological activity. The compounds 5‐{1‐(4‐Chlorophenyl)‐3‐[4‐(methoxy‐phenyl)‐prop‐2‐en‐1‐ylidene]amino}‐1,3,4‐thiadiazole‐2‐thiol ( 2b ), 5‐{[1‐(4‐chloro‐phenyl)]‐3‐[4‐(dimethyl‐amino‐phenyl)‐prop‐2‐en‐1‐ylidene]amino}‐1,3,4‐thiadiazole‐2‐thiol ( 2c ) and 5‐{[1‐(4‐chloro‐phenyl)]‐3‐[(4‐amino‐phenyl)‐prop‐2‐en‐1‐ylidene]amino}‐1,3,4‐thiadiazole‐2‐thiol ( 2f ) showed 100% activity in comparison with standard Acetazolamide, a known anti‐convulsant drug. The compounds 2c , 2f also passed the Rotarod and Ethanol Potentiation tests which further confirmed them to be safe in motor coordination activity and safe from generating neurological toxicity.